An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor

Abstract

The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure of the Y2H depends primarily on the ability to avoid false-negative and false-positive hits that become a limiting factor for the value of the system, especially in large scale proteomic analyses. We provide here a systematic assessment of parameters to help improving the quality of split-ubiquitin cDNA-library screenings. We experimentally defined the optimal 5-fluoroorotic acid (5-FOA) concentration as a key parameter to increase the reproducibility of interactions and, at the same time, to keep non-specific background growth low. Furthermore, we show that the efficacy of the 5-FOA selection is modulated by the plating density of the yeast clones. Moreover, a reporter-specific class of false-positive hits was identified, and a simple phenotypic assay for efficient de-selection was developed. We demonstrate the application of this improved system to identify novel interacting proteins of the human Frizzled 1 receptor. We identified several novel interactors with components of the Wnt-Frizzled signalling pathways and discuss their potential roles as direct mediators of Frizzled receptor signalling. The present work is the first example of a split-ubiquitin interaction screen using an in-situ expressed receptor of the serpentine class, emphasizing the suitability of the described improvements in the screening protocol.

New Perspectives in Predicting Membrane Protein-Protein Interactions

“…Although there is no evidence demonstrating the direct interaction between NUP116 and ATP14, some research results indicate that ATP14 might be involved in ATP synthesis in the process of protein importing into nucleus (Dingwall & Laskey, 1986;Vargas et al, 2005). Interestingly, we found some new complexes such as peroxisome organization and biogenesis related to the functions of peroxisome membrane proteins such as peroxisome biogenesis and peroxisomal matrix protein import (Eckert & Erdmann, 2003;Heiland & Erdmann, 2005;Honsho et al, 2002).…”

“…Recently, more applications of the split-ubiquitin approach have been proposed. For example, novel interactors of the yeast ABC transporter Ycf1p (Paumi et al, 2007) and the human Frizzled 1 receptor (Dirnberger et al, 2008) have been identified using such method.…”

Techniques for the analysis of protein-protein interactions in vivo

“…Unbiased approaches to obtain novel interactors of a POI include cDNA library screens using the yeast two-hybrid system (Gietz, 2006), the split-ubiquitin system (Dirnberger et al, 2008), and the (now more affordable) CoIP-MS technique (Pertl-Obermeyer et al, 2014). It is imperative, however, to critically test an interaction couple individually and independently (with alternative techniques) before investing too much time and effort in analysis.…”

“…The possibility of analyzing membrane proteins as potential enhancer, bridging, or inhibiting factors becomes increasingly interesting, for example, in the analysis of (ligand-induced) receptor oligomerization. Apoplastic proteins, peptides, small molecules, or drugs can be screened for their potential to inhibit or increase the interactions of membranebound receptors (Dirnberger et al, 2008;Grefen, 2014;Wehr and Rossner, 2016).…”

AIP and its interacting partners

“…In order to identify novel signalling pathways induced by RET51, Vargiolu et al (2009) used the new split-ubiquitin Y2H assay (Dirnberger et al 2008) to screen a human fetal cDNA library. Among the ten clones identified, one corresponded to AIP.…”